Peptides with anti-aging activity and uses thereof

Abstract

The present invention relates to a peptide having an anti-aging activity, and a composition for inhibiting skin aging or regenerating skin, which comprises the peptide as an active ingredient.

Description

Technical Field

[0001] This invention relates to peptides with anti-aging activity, and compositions containing the peptide as an active ingredient for inhibiting skin aging or regenerating skin. Background Technology

[0002] Skin cells age due to various reasons, and wrinkles result from this aging. Aging can be divided into age-related aging (intrinsic aging) and photoaging (photochemical aging). Age-related aging is the natural aging process that occurs over time based on biological processes, while photoaging involves degenerative changes in areas exposed to sunlight. Photoaging and age-related aging can occur in combination. Natural cellular aging can occur when the expression or activity of various factors related to cell growth is inhibited, and skin cells can undergo photoaging due to light with wavelengths from 280 nm to 400 nm contained in sunlight. In particular, exposure to ultraviolet radiation such as UVB with wavelengths in the range of 280 nm to 320 nm can damage the skin or fibers and cause tanning, thus darkening the skin tone. When UVB irradiates skin cells, it promotes the accumulation of ROS and free radicals in the skin cells and stimulates intracellular signal transduction systems induced by free radicals. This can induce oxidative stress in biomolecules such as DNA, proteins, and lipids, thus causing damage to skin tissue.

[0003] When oxidative stress in skin cells increases, keratinocytes in the epidermis or fibroblasts in the dermis are stimulated. The expression of genes acting as collagen-degrading enzymes, such as matrix metalloproteinases (MMPs), increases through a series of intracellular signal transduction processes, leading to a reduction in collagen. Collagen, a major component of the skin (comprising 90% of the dermis), protects the skin from external stimuli and forces by providing strength and tension. This results in skin aging or wrinkles. Therefore, regulating the expression of genes involved in the synthesis or degradation of fibroblasts such as collagen is expected to prevent skin cell aging and reduce wrinkles.

[0004] The cellular aging process caused by ultraviolet radiation from sunlight not only leads to cosmetic problems due to skin damage, but also to serious health problems, such as cancer caused by DNA damage to cells, damage to the central and peripheral nervous systems, disruption of the immune system, reproductive organ abnormalities, developmental abnormalities in infants and young children, and skin diseases such as chloracne. Therefore, it is urgent to address these issues.

[0005] To address these issues, various materials have been developed, and attempts have been made to overcome them using extracts obtained from various plants or microorganisms. However, in many cases, the exact composition of the extracts is unknown because it is impossible to specifically identify which substances in extracts obtained from natural products are effective in protecting skin cells or reversing signs of aging. Therefore, extract components containing unknown substances have also been used. Given that these substances will be in direct contact with or applied to the human body, their side effects should be minimized, and human safety must be ensured. Therefore, there is a need to accurately identify single substances with the effects that can address the aforementioned problems and apply them to human skin cells.

[0006] [Existing Technical Documents]

[0007] [Non-patent literature] S Edgar et al., Effects of collagen-derived bioactivepeptides and natural antioxidant compounds on proliferation and matrixprotein synthesis by cultured normal human dermal fibroblasts, Scientific Reports, 2018; 8:10474 Summary of the Invention

[0008] Technical issues

[0009] The purpose of this invention is to provide a peptide with anti-aging activity.

[0010] Another object of the present invention is to provide a composition for inhibiting skin aging or regenerating skin, comprising a peptide having anti-aging activity as an active ingredient.

[0011] Another object of the present invention is to provide a cosmetic composition for inhibiting skin aging or regenerating skin, comprising a peptide having anti-aging activity as an active ingredient.

[0012] Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of photoaging, comprising a peptide having anti-aging activity as an active ingredient.

[0013] Technical solution

[0014] To achieve the above objectives, one aspect of the present invention provides a peptide comprising the amino acid sequence of SEQ ID NO: 1.

[0015] Another aspect of the present invention provides a composition for inhibiting skin aging or regenerating skin, comprising a peptide having anti-aging activity as an active ingredient.

[0016] Another aspect of the present invention provides a cosmetic composition for inhibiting skin aging or regenerating skin, comprising a peptide having anti-aging activity as an active ingredient.

[0017] Another aspect of the present invention provides a pharmaceutical composition for the prevention or treatment of photoaging, comprising a peptide having anti-aging activity as an active ingredient.

[0018] The present invention will be described in detail below.

[0019] One aspect of the present invention provides a peptide comprising the amino acid sequence described in SEQ ID NO: 1.

[0020] In this specification, the term "peptide" refers to a linear molecule composed of amino acid residues, and more specifically, the peptides of the present invention may include the amino acid sequence described in SEQ ID NO: 1.

[0021] Within the scope of not affecting functionality, a "peptide" can be a variant or fragment of amino acids that have a different sequence through the deletion, insertion, substitution, or combination of amino acid residues. Amino acid exchanges that do not completely alter the activity of the peptide are known in the art. In some cases, peptides can be modified by phosphorylation, sulfation, acrylation, glycosylation, methylation, farnesylation, etc. Therefore, the peptides of the present invention include peptides containing an amino acid sequence substantially identical to that of a peptide comprising the amino acid sequence of SEQ ID NO: 1, its variants, or its active fragment.

[0022] The term "essentially identical protein" refers to an amino acid sequence that has 75% or more, preferably 80% or more, such as 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more of the amino acid sequence of SEQ ID NO: 1. However, it is not limited to this. As long as the protein has 75% or more of the amino acid sequence homology and has the same activity, it is included within the scope of this invention.

[0023] Furthermore, the peptides of the present invention may also include targeting sequences, tags, labeled residues, or amino acid sequences prepared for a specific purpose to increase the half-life or stability of the peptide.

[0024] Furthermore, to obtain better chemical stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc.), altered specificity (e.g., a broad bioactivity spectrum), and reduced antigenicity, the N-terminus or C-terminus of the peptides of the present invention may be modified. "Stability" includes not only in vivo stability for protecting the peptides of the present invention from attack by proteases in vivo, but also storage stability (e.g., storage stability at room temperature).

[0025] N-terminal modification can be, but is not limited to, binding of a protecting group selected from the group consisting of acetyl, fluorenylmethoxycarbonyl, formyl, palmitoyl, myristoyl, stearoyl, and polyethylene glycol (PEG) to the N-terminus of the peptide.

[0026] The C-terminus can be modified by the binding of hydroxyl (-OH), amino (-NH2), hydrazine (-NHNH2), etc., to the C-terminus of the peptide, but is not limited to these.

[0027] The peptides of this invention can be synthesized, for example, using apparatus or genetic engineering techniques. When synthesizing peptides using an apparatus, the desired peptides can be synthesized using the Fmoc solid-phase method in an automated peptide synthesizer.

[0028] In a specific embodiment of the present invention, the peptide comprising the amino acid sequence of SEQ ID NO: 1 of the present invention is synthesized and identified using the Fmoc solid-phase method, and selection is made by verifying the efficacy of the identified peptide.

[0029] In a specific embodiment of the present invention, in order to verify the efficacy of the identified peptide, the peptide of the present invention is selected by verifying the following effects of the peptide: the effect of increasing the expression levels of extracellular matrix (ECM) components and skin barrier strengthening factors, the effect of reducing the level of reactive oxygen species increased by ultraviolet radiation, and the effect of inhibiting the expression and activity of collagen degradation-related proteins increased by ultraviolet radiation or heat treatment.

[0030] Therefore, the peptide of the present invention containing the amino acid sequence of SEQ ID NO: 1 has skin aging inhibitory activity or skin regeneration activity.

[0031] Another aspect of the present invention provides a composition for inhibiting skin aging or regenerating skin, having a peptide comprising the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0032] Skin aging can be the natural aging of cells over time, or it can be photoaging caused by sunlight. Furthermore, aging can be induced by intracellular oxidative stress, which can be caused by various factors. Therefore, cell growth inhibition or apoptosis may occur, the synthesis of various fibrous proteins that make up skin cells may be suppressed, and the expression of degradative enzymes may increase. In particular, when exposed to ultraviolet radiation, the expression of genes such as Col1a1, fibronectin, and elastin in skin cells is suppressed, which can lead to photoaging and wrinkles.

[0033] "Skin regeneration" can promote the growth and vitality of skin cells to prevent damage to cells from external and / or internal stimuli, reduce skin wrinkles, improve elasticity, and strengthen the skin barrier. Skin regeneration also includes preventing skin damage caused by external environmental factors (such as pigmentation) or improving or treating photoaging of the skin.

[0034] In one embodiment, the skin aging inhibition or skin regeneration composition enhances resistance to cell damage caused by aging or external stimuli.

[0035] "External stimuli" can be physical stimuli, chemical stimuli caused by the application of cosmetics or other external preparations, stimuli caused by ultraviolet rays, or stimuli caused by infrared rays.

[0036] Damaged cells can be skin fibroblasts or keratinocytes.

[0037] The peptide of this invention can induce an increase in the expression of fibrin synthesis genes in skin cells. Therefore, this peptide can reduce wrinkles and improve skin elasticity by increasing fibrin synthesis, and can also increase the resistance and vitality of skin cells by improving the skin barrier, and enhance the regenerative capacity of skin damaged by external stimuli or aging. The skin aging inhibition and skin regeneration effects of the peptide of this invention can be confirmed by measuring the expression levels of proteins involved in cell proliferation or anti-aging genes, as well as fibrin, whose expression levels change within skin cells as skin aging progresses.

[0038] In one embodiment, the skin aging inhibition or skin regeneration composition can increase the expression or secretion of collagen, fibronectin, elastin or hyaluronic acid in skin cells.

[0039] In one embodiment, the composition for inhibiting skin aging or regenerating skin can increase the expression of SIRT1 or AQP3, which are skin barrier strengthening factors.

[0040] In one embodiment, the skin aging inhibition or skin regeneration composition can inhibit the production of reactive oxygen species (ROS) caused by ultraviolet radiation.

[0041] In one embodiment, the skin aging inhibition or skin regeneration composition can increase the expression of collagen, fibronectin, or elastin that is reduced by ultraviolet radiation.

[0042] In one embodiment, the skin aging inhibition or skin regeneration composition can inhibit the expression of MMP-1 or the activity of MMP-2 increased by ultraviolet light or heat treatment.

[0043] In a specific embodiment of the invention, it is shown that the peptide of the present invention comprising the amino acid sequence of SEQ ID NO: 1 increases the expression of genes encoding extracellular matrix proteins such as fibronectin and elastin in fibroblasts.

[0044] Furthermore, it has been shown that this peptide increases the secretion of collagen or hyaluronic acid by fibroblasts or keratinocytes.

[0045] Furthermore, it was shown that this peptide increases the expression of skin barrier strengthening genes such as SIRT1 and AQP3 in keratinocytes.

[0046] Furthermore, the peptide was shown to reduce the level of intracellular reactive oxygen species (ROS) in fibroblasts damaged by ultraviolet radiation.

[0047] Furthermore, it was shown that the peptide increased the expression of Col1a1, fibronectin, and elastin genes, which were reduced in fibroblasts damaged by ultraviolet radiation.

[0048] Furthermore, it was shown that the peptide inhibited the increased expression of MMP-1 or the activity of MMP-2 in fibroblasts damaged by ultraviolet radiation.

[0049] Furthermore, it was shown that this peptide inhibited the increased expression of MMP-1 in fibroblasts damaged by heat treatment.

[0050] Therefore, it is obvious that the peptides of the present invention have skin aging inhibition or skin regeneration activity by regulating the expression of genes related to skin cell aging, and the peptides of the present invention can be effectively used as active ingredients in compositions for skin aging inhibition or skin regeneration.

[0051] Another aspect of the present invention provides a cosmetic composition for inhibiting skin aging or regenerating skin, having a peptide comprising the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0052] In one implementation, skin aging inhibition or skin regeneration can prevent or reduce skin wrinkles, improve skin elasticity, strengthen the skin barrier, prevent pigmentation, or improve photoaging of the skin.

[0053] The peptide containing the amino acid sequence of SEQ ID NO: 1 is the same as the peptide described in "Composition for Inhibiting or Regenerating Skin Aging," and therefore its detailed description is provided above. Hereinafter, only the specific formulation of the cosmetic composition for inhibiting or regenerating skin aging will be described.

[0054] Cosmetic compositions can be prepared into any formulation commonly prepared in this field, such as solutions, suspensions, lotions, creams, gels, lotions, toners, powders, soaps, surfactant-containing cleansers, oils, powder foundations, lotion foundations, wax foundations, and sprays, but are not limited thereto.

[0055] Cosmetic compositions can be prepared in various forms, such as solutions, sols and gels, emulsions, oils, waxes, and aerosols, including, but not limited to, toners, nourishing lotions, nourishing creams, massage creams, serums, eye creams, cleansing creams, cleansing foams, cleansing waters, face masks (packs), sprays, powders, hair growth creams, hair pastes, hair conditioners, shampoos, hair conditioners, hair gels, hair aerosols, hair oils, gels, etc.

[0056] The cosmetic compositions of the present invention may contain other additives, such as excipients and carriers, and can be applied and mixed as needed with commonly used ingredients that can be used with ordinary skin cosmetics.

[0057] When the cosmetic composition of the present invention is formulated as a cream, lotion, or gel, animal oil, vegetable oil, wax, paraffin, starch, tragacanth gum, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, talc, or zinc oxide may be used as a carrier component.

[0058] When the cosmetic composition is formulated as a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder may be used as a carrier component, and in particular, when the cosmetic composition is formulated as a spray, it may additionally contain a propellant such as chlorofluorocarbon, propane / butane, or dimethyl ether, but is not limited thereto.

[0059] When the cosmetic composition is formulated as a solution or emulsion, a solvent, solubilizer, or emulsifier may be used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butanediol oil, glycerol aliphatic esters, polyethylene glycol, fatty acid esters of sorbitol, etc.

[0060] When the cosmetic composition is formulated as a suspension, liquid diluents such as water, ethanol, or propylene glycol can be used, and suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitan ester or polyoxyethylene dehydrated sorbitan ester, microcrystalline cellulose, aluminum hydroxide, bentonite, agar, tragacanth gum, etc.

[0061] When the cosmetic composition is formulated as a detergent containing surfactants, fatty alcohol sulfates, fatty alcohol ether sulfates, sulfosuccinate monoesters, hydroxyethyl sulfonates, imidazolonium derivatives, methyl taurine, sarcosinates, fatty acid amide ether sulfates, alkylamide betaine, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives, ethoxylated glycerol fatty acid esters, etc., can be used as carrier components.

[0062] When the cosmetic composition is formulated as a shampoo, basic ingredients used to form the shampoo, such as thickeners, surfactants, viscosity modifiers, humectants, pH adjusters, preservatives, or essential oils, can be mixed with the peptides of the present invention. CDE can be used as a thickener, LES as an anionic surfactant or cocobetaine as an amphoteric surfactant can be used as a surfactant, polyquaternium salts can be used as viscosity modifiers, glycerin can be used as a humectant, and citric acid or sodium hydroxide can be used as a pH adjuster. Grapefruit extract and the like can be used as preservatives, and essential oils such as cedarwood, peppermint, or rosemary, serine amino acids, pentanol, or vitamin E can also be added.

[0063] In addition to the peptides and carrier components of the present invention, the cosmetic composition may also contain ingredients commonly used in cosmetic compositions, such as conventional adjuvants, antioxidants, stabilizers, solubilizers, vitamins, pigments and fragrances as active ingredients, but is not limited thereto.

[0064] Another aspect of the present invention provides a pharmaceutical composition for the prevention or treatment of photoaging having a peptide comprising the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0065] The peptide containing the amino acid sequence of SEQ ID NO: 1 is the same as the peptide described in "Compositions for Inhibiting or Regenerating Skin Aging," and therefore its detailed description is provided above. Hereinafter, only the specific formulation of the pharmaceutical composition for preventing or treating photoaging will be described.

[0066] In this specification, "photoaging" refers to skin damage, pigmentation, wrinkles, or decreased skin elasticity caused by repeated or prolonged exposure to sunlight.

[0067] In this specification, "prevention" means any action by which the peptide of the present invention or a composition containing the peptide inhibits or delays the occurrence, spread, and recurrence of photoaging.

[0068] In this specification, "improvement or treatment" means any beneficial alteration of photoaging behavior, such as delaying, stopping, or reversing the progression of photoaging by means of the peptide of the present invention or a composition containing the peptide.

[0069] In a specific embodiment of the invention, the peptide increases the expression or secretion of extracellular matrix (ECM) proteins such as collagen, fibronectin, elastin and hyaluronic acid, and increases the expression of skin barrier strengthening factors such as SIRT1 or AQP3, thereby preventing, improving or treating photoaging.

[0070] In addition, this peptide inhibits the production of reactive oxygen species (ROS) in cells damaged by aging or external stimuli, increases the expression of decreased collagen, fibronectin or elastin, and inhibits the increased expression of MMP-1 or the elevated activity of MMP-2, thereby preventing, improving or treating photoaging.

[0071] The pharmaceutical compositions of the present invention may also contain a pharmaceutically acceptable carrier.

[0072] The pharmaceutical composition may also contain, for example, a carrier for oral administration or a carrier for parenteral administration as a pharmaceutically acceptable carrier. Carriers for oral administration may include lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, etc. Furthermore, carriers for parenteral administration may include water, suitable oils, salt solutions, aqueous glucose solutions, ethylene glycol, etc. In addition, the pharmaceutical composition may contain stabilizers and preservatives. Suitable stabilizers include antioxidants such as sodium bisulfite, sodium sulfite, and ascorbic acid. Suitable preservatives include benzalkonium chloride, methylparaben or propylparaben, and chlorobutanol. For a description of suitable pharmaceutically acceptable carriers, refer to Remington's Pharmaceutical Sciences (19th edition, Mack Publishing Company, Easton, PA, 1995).

[0073] The pharmaceutical compositions of the present invention can be administered to mammals, including humans, by any method. For example, the pharmaceutical compositions of the present invention can be administered orally or parenterally. Parenterally administration can be (but is not limited to) intravenous, intramuscular, intra-arterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, local, sublingual, or rectal administration.

[0074] The pharmaceutical compositions of the present invention can be formulated into oral or parenteral formulations according to the administration routes described above, and the parenteral formulations can be specifically formulated and used in the form of injectable or topical formulations.

[0075] The pharmaceutical compositions of the present invention are administered in pharmaceutically effective amounts. In this invention, a "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio suitable for medical treatment. Effective dosage levels can be determined based on factors including the patient's disease type and severity, drug activity, drug sensitivity, timing of administration, route of administration, excretion rate, duration of treatment, and concurrently used drugs, as well as other factors known in the medical field. The pharmaceutical compositions can be administered as a single therapeutic agent or in combination with other therapeutic agents; the pharmaceutical compositions can be administered simultaneously, separately, or sequentially with conventional therapeutic agents; or the pharmaceutical compositions can be administered alone or multiple times. Importantly, considering all the foregoing factors, the pharmaceutical composition can be administered in an amount that yields maximum effect with minimal dosage and without side effects, and the amount of pharmaceutical composition to be administered can be readily determined by those skilled in the art.

[0076] The effective amount of the drug composition can vary depending on the patient's age, sex, condition, weight, the extent of absorption, inactivation rate, excretion rate of the active ingredient in the body, disease type, and concomitant drugs. Furthermore, the effective amount of the drug composition can be increased or decreased depending on the route of administration, severity, sex, weight, age, etc. For example, the drug composition can be administered at a dose of approximately 0.0001 μg to 500 mg per kg of patient body weight per day, preferably 0.01 μg to 100 mg.

[0077] As described above, the peptides of the present invention have excellent skin aging inhibition and skin regeneration effects, and therefore can be used as raw materials for cosmetics for skin aging inhibition or skin regeneration, or as raw materials for pharmaceutical products for preventing, improving or treating photoaging.

[0078] Beneficial effects

[0079] The peptide containing the amino acid sequence of SEQ ID NO: 1 according to the present invention has anti-aging activity and can therefore be used as a raw material for cosmetics that inhibit skin aging or regenerate skin, or as a raw material for pharmaceutical products that prevent, improve or treat photoaging.

[0080] The effects of the present invention are not limited to those described above, and other effects not mentioned can be clearly understood by those skilled in the art from the following description. Attached Figure Description

[0081] Figure 1 The figure illustrates the effect of treating fibroblasts with the peptides of the present invention on the expression of fibronectin and elastin, where Con represents the untreated group and bFGF represents the positive control.

[0082] Figure 2The figure illustrates the effect of treating fibroblasts (A) and keratinocytes (B) with the peptides of the present invention on the secretion of collagen and hyaluronic acid, where Con represents the untreated group and TGF-β1 and IGF represent positive controls.

[0083] Figure 3 The figure illustrates the effect of treating keratinocytes with the peptides of the present invention on the expression of SIRT1 and AQP3, where Con represents the untreated group and EGF represents the positive control.

[0084] Figure 4 The figure illustrates the effect of the peptides of the present invention on the increased levels of intracellular reactive oxygen species (ROS) in keratinocytes due to ultraviolet (UV) irradiation, where Con represents the non-UV irradiation group, NC represents the UV irradiation group, 10 μM, 50 μM and 100 μM represent groups irradiated with UV and treated with different concentrations of the peptides of the present invention, and NaC represents the positive control irradiated with UV and treated with N-acetylcysteine.

[0085] Figure 5 The figure illustrates the effect of the peptides of the present invention on the expression of Col1a1, fibronectin, and elastin genes in fibroblasts that are reduced by ultraviolet (UV) irradiation. In this figure, Con represents the non-UV irradiation group, NC represents the UV irradiation group, 10 μM, 50 μM, and 100 μM represent groups irradiated with UV and treated with different concentrations of the peptides of the present invention, and quercetin represents the positive control irradiated with UV and treated with quercetin.

[0086] Figure 6 The figure illustrates the effect of the peptides of the present invention on the increased expression of MMP-1 and the activity of MMP-2 in fibroblasts due to ultraviolet (UV) irradiation, where Con represents the non-UV irradiation group, NC represents the UV irradiation group, 10 μM, 50 μM and 100 μM represent groups irradiated with UV and treated with different concentrations of the peptides of the present invention, and NaC represents the positive control irradiated with UV and treated with N-acetylcysteine.

[0087] Figure 7 The figure illustrates the effect of the peptide of the present invention on the expression of MMP-1 increased by heat treatment in fibroblasts, where Con represents the non-UV irradiation group, NC represents the UV irradiation group, 10 μM, 50 μM and 100 μM represent groups irradiated with ultraviolet light and treated with different concentrations of the peptide of the present invention, and NaC represents the positive control irradiated with ultraviolet light and treated with N-acetylcysteine. Detailed Implementation

[0088] The present invention will be described in detail below with reference to embodiments.

[0089] However, the following embodiments are merely illustrative of the present invention, and the present invention is not limited to the following embodiments.

[0090] example

[0091] [Synthesis example 1]

[0092] Synthesis of a peptide containing the amino acid sequence of SEQ ID NO:1

[0093] Peptides containing the amino acid sequence of SEQ ID NO:1 [Table 1] were synthesized using an automated peptide synthesizer (Liberty, CEM Corporation, USA), and then the synthesized peptides were separated and purified using C18 reversed-phase high-performance liquid chromatography (HPLC) (U-3000, Thermofisher Scientific, USA). Pursuit XRs C18 (250...) (4.65 mm 100 Å, Agilent, USA) was used as a chromatographic column.

[0094] [Table 1]

[0095] [Experimental Example 1] Confirmation of the inhibitory effect of peptide treatment on natural aging

[0096] 1-1. Confirmation of increased expression of extracellular matrix (ECM) component genes (collagen, fibronectin, and elastin)

[0097] The expression levels of genes involved in the natural aging of fibroblasts were measured to confirm whether the peptides of the present invention inhibited the naturally occurring cellular aging phenomenon.

[0098] First, NIH3T3 cells (mouse fibroblast line) were thromboplasted at a rate of 3 × 10⁻⁶. 5 Cells were seeded at a density of [number] cells / well in 6-well plates and then cultured in DMEM medium containing 10% fetal bovine serum (FBS) for 24 hours. The cultured cells were washed once with serum-free DMEM medium, and the peptides of the present invention were mixed into 1 mL of serum-free DMEM medium at concentrations of 10 µM, 50 µM, and 100 µM, respectively, and dispensed into the cells. Positive controls (PCs) were treated with 5 ng / mL TGF-β1 instead of the peptides. Cells were cultured in a CO2 incubator at 37°C for 24 hours.

[0099] The cultured cells were washed twice with PBS, and RNA was extracted from the cells using Easy Blue (iNtRON, Cat. No.: 17061, Korea). The amount of extracted RNA was quantified by adding 1,000 ng of RNA to each tube and performing an RT reaction using a kit (Enzynomics, Cat. No.: PT200, Korea). RT-PCR was performed using a PCR kit (Enzynomics, Cat. No.: P581T, Korea). The sequences of the primers for fibronectin and elastin used in RT-PCR, as well as the sequences of the primers for GAPDH used to compare the total RNA amount in each treatment group, are shown in [Table 2].

[0100] [Table 2]

[0101] As a result, such Figure 1 As shown, it was confirmed that when treated with peptides, the expression of fibronectin and elastin genes gradually increased with increasing peptide concentration. Therefore, it can be concluded that the peptide containing the amino acid sequence of SEQ ID NO: 1 increases the expression of fibronectin and elastin, which are extracellular matrix proteins constituting dermal fibroblasts, thus achieving an effect of inhibiting or improving natural aging such as skin wrinkles.

[0102] 1-2. Confirmation of increased protein secretion of extracellular matrix (ECM) components (collagen and hyaluronic acid)

[0103] The secretion levels of proteins involved in the natural aging of fibroblasts and keratinocytes were measured to confirm whether the peptides of the present invention inhibited the naturally occurring cellular aging process.

[0104] First, NIH3T3 cells and HaCaT cells (human keratinocyte line) were mixed at a ratio of 1×10⁻⁶. 4 Cells were seeded at a density of 10 cells / well in 24-well plates and then cultured in DMEM medium containing 10% fetal bovine serum (FBS) for 24 hours. The cultured cells were washed once with serum-free DMEM medium, and the peptides of the present invention were mixed in 2 mL of serum-free DMEM medium at concentrations of 10 µM, 50 µM, 100 µM, and 5 ng / mL, respectively, and dispensed into three wells at 500 µL per condition. NIH3T3 cells were treated with 5 ng / mL TGF-β1 instead of the above peptides as a positive control (PC). HaCaT cells were treated with 10 nM IGF-1 instead of the above peptides as a positive control (PC). Cells were cultured in a CO2 incubator at 37°C for 72 hours.

[0105] The culture medium of NIH3T3 and HaCaT cells was centrifuged to allow the suspension to settle, and the supernatant was transferred to e-tubes. Then, ELISA (Abcam, Cat. No.: ab210579, UK) and hyaluronic acid (HA) ELISA (ECHELON biosciences, Cat. No.: K-1200, USA) were performed using the same method.

[0106] As a result, such Figure 2 As shown, it was confirmed that when treated with peptides, the secretion of collagen and hyaluronic acid gradually increased with increasing peptide concentration.

[0107] It can be concluded that the peptide containing the amino acid sequence of SEQ ID NO: 1 increases the amount of collagen and hyaluronic acid proteins, which are extracellular matrix proteins that make up dermal fibroblasts and keratinocytes, thus achieving the effect of inhibiting or improving natural aging such as skin wrinkles.

[0108] 1-3. Confirmation of increased expression of skin barrier function-related genes (SIRT1 and AQP3)

[0109] The expression levels of genes involved in the natural aging of keratinocytes were measured to confirm whether the peptides of the present invention inhibited the naturally occurring cellular aging process.

[0110] First, HaCaT cells were loaded at a rate of 3 × 10⁻⁶. 5 Cells were seeded at a density of [number] cells / well in 6-well plates and then cultured in DMEM medium containing 10% fetal bovine serum (FBS) for 24 hours. The cultured cells were washed once with serum-free DMEM medium, and then the peptides of the present invention were mixed at concentrations of 10 µM, 50 µM, and 100 µM in 1 mL of serum-free DMEM medium and dispensed into the cells. Positive controls (PCs) were treated with 100 nM EGF instead of the peptides. Cells were cultured in a CO2 incubator at 37°C for 24 hours.

[0111] The cultured cells were washed twice with PBS, and RNA was then extracted from the cells using Easy Blue (iNtRON, Cat. No.: 17061, Korea). The extracted RNA was subjected to an RT reaction using a kit (Enzynomics, Cat. No.: RT200, Korea). Subsequently, RT-PCR was performed using a PCR kit (Enzynomics, Cat. No.: R581T, Korea). The sequences of the primers for SIRT1 and AQP3 used in RT-PCR, as well as the sequences of the primers for GAPDH used to compare the total RNA amount in each treatment group, are shown in [Table 2].

[0112] As a result, such Figure 3 As shown, it was confirmed that when treated with peptides, the mRNA levels of SIRT1 and AQP3, which are skin barrier strengthening factors, increased.

[0113] Therefore, it can be concluded that the peptide containing the amino acid sequence of SEQ ID NO: 1 increases the expression levels of SIRT1 and AQP3, which are skin barrier strengthening factors in keratinocytes, thus achieving the effect of inhibiting or improving natural aging.

[0114] [Experimental Example 2]

[0115] Confirmation of the inhibitory effect of peptide treatment on extrinsic aging

[0116] 2-1. Confirmation of the effect of inhibiting reactive oxygen species increased by ultraviolet radiation.

[0117] UV irradiation increases the level of reactive oxygen species (ROS). Therefore, it was confirmed whether treatment with the peptides of the present invention reduced ROS levels again and whether it inhibited aging caused by oxidative stress in cells.

[0118] First, NIH3T3 cells were loaded at 5 × 10⁻⁶. 5 Cells were seeded at a density of 10 cells / well in 6-well plates and then cultured in DMEM medium containing 10% fetal bovine serum (FBS) for 24 hours. The cultured cells were washed once with serum-free DMEM medium, and then the peptides of the present invention were mixed in 1 mL of serum-free DMEM medium at concentrations of 10 µM, 50 µM, and 100 µM, respectively, and dispensed into the cells. Positive controls (PCs) were treated with 7.5 mM N-acetylcysteine ​​(NaC) instead of the peptides.

[0119] Cells were cultured in a CO2 incubator at 37°C for 1 hour. The culture medium was transferred to tubes, 1 ml of PBS was added, and then the cells were irradiated with a UV irradiator (VILBER LOURMAT, Cat No.: 3102-BSU, France) at 8 J / cm². 2 Cells were irradiated with UVA. After removing PBS, 900 µL of the culture medium was transferred to tubes and incubated in a CO2 incubator at 37°C for 24 hours. After treatment with 10 µM dichlorodihydrofluorescein diacetate (DCFH-DA), cells were wrapped in foil and incubated in a CO2 incubator at 37°C for 30 minutes. Cells were washed twice with PBS and 500 μL of 1XTE was dispensed to separate the cells. After centrifugation, cells were washed with PBS and fluorescence values ​​were measured by FACS.

[0120] As a result, such Figure 4 As shown, it was confirmed that when NIH3T3 cells were treated with the peptides of the present invention, the level of intracellular reactive oxygen species increased by ultraviolet irradiation was reduced.

[0121] Therefore, it can be concluded that the peptide containing the amino acid sequence of SEQ ID NO: 1 reduces the level of reactive oxygen species in keratinocytes that are increased by UV irradiation and inhibits oxidative stress, thus preventing photoaging of cells.

[0122] 2-2. Confirmation of increased expression of genes for extracellular matrix components (collagen, fibronectin, and hyaluronic acid) that are inhibited by ultraviolet radiation.

[0123] UV irradiation inhibits the expression of genes related to extracellular matrix components. Therefore, by confirming whether the expression levels of Col1a1, fibronectin, and elastin genes recovered and increased again, it was confirmed that photoaging of cells was inhibited when treated with the peptides of the present invention.

[0124] First, NIH3T3 cells were loaded at 5 × 10⁻⁶. 5 Cells were seeded at a density of [number] cells / well in 6-well plates and then cultured in DMEM medium containing 10% fetal bovine serum (FBS) for 24 hours. The cultured cells were washed once with serum-free DMEM medium, and then the peptides of the present invention were mixed in 1 mL of serum-free DMEM medium at concentrations of 10 µM, 50 µM, and 100 µM, respectively, and dispensed into the cells. The positive control (PC) was treated with 50 µM quercetin instead of the peptides. The cells were cultured in a CO2 incubator at 37°C for 6 hours.

[0125] Transfer the culture medium to an e-tube, add 1 ml of PBS, and incubate using a UV irradiator (VILBER LOURMAT, Cat No.: 3102-BSU, France) at 6 J / cm². 2The cells were irradiated with UVA. After removing PBS, 900 µL of culture medium was transferred to tubes and incubated in a CO2 incubator at 37°C for 6 hours. The cultured cells were washed twice with PBS, and RNA was extracted from the cells using Easy Blue (iNtRON, Cat. No.: 17061, Korea). The amount of extracted RNA was quantified by adding 1,000 ng of RNA to each tube and performing an RT reaction using a kit (Enzynomics, Cat. No.: RT200, Korea). Subsequently, RT-PCR was performed using a PCR kit (Enzynomics, Cat. No.: R581T, Korea). The sequences of primers for Col1a1, fibronectin, and elastin used in RT-PCR, as well as the sequences of primers for GAPDH used to compare the total RNA amount in each treatment group, are shown in [Table 2].

[0126] As a result, such Figure 5 As shown, it was confirmed that when treated with the peptides of the present invention, the mRNA levels of Col1a1, fibronectin, and elastin, which were reduced by ultraviolet irradiation, increased again.

[0127] Therefore, it can be concluded that the peptide containing the amino acid sequence of SEQ ID NO: 1 restores the expression levels of Col1a1, fibronectin, and elastin in fibroblasts that were reduced by UV irradiation, thus inhibiting the photoaging of cells.

[0128] 2-3. Confirmation of inhibition of UV-induced increased MMP-1 expression and MMP-2 activity

[0129] Ultraviolet radiation increases the expression of MMP-1 and MMP-2 proteins, which are associated with collagen degradation. Therefore, it was confirmed whether treatment with the peptides of the present invention re-inhibited the synthesis of MMP-1 and MMP-2 proteins, and whether cellular senescence was inhibited.

[0130] First, NIH3T3 cells were loaded at 5 × 10⁻⁶. 5 Cells were seeded at a density of 10 cells / well in 6-well plates and then cultured in DMEM medium containing 10% fetal bovine serum (FBS) for 24 hours. The cultured cells were washed once with serum-free DMEM medium, and the peptides of the present invention were mixed in 1 mL of serum-free DMEM medium at concentrations of 10 µM, 50 µM, and 100 µM, respectively, and dispensed into the cells. Positive controls (PCs) were treated with 2.5 mM N-acetylcysteine ​​(NaC) instead of the peptides.

[0131] Cells were cultured in a CO2 incubator at 37°C for 1 hour. The culture medium was transferred to tubes (e-tubes), 1 ml of PBS was added, and then the cells were irradiated with UV light (VILBER LOURMAT, Cat No.: 3102-BSU, France) at 6 J / cm². 2 The cells were irradiated with UVA. After removing the PBS, 900 µL of culture medium was added to the tubes and the cells were incubated in a CO2 incubator at 37°C for 6 hours. The culture medium was transferred to tubes, the cells were washed twice with PBS, and then cell lysis buffer was added to lyse the cells. Samples were prepared by treating the cells with 5X sample buffer and then performing SDS-PAGE on a 10% SDS-PAGE gel. The proteins separated by SDS-PAGE were transferred to a PVDF membrane. The membrane was blocked with 5% skim milk at room temperature for 1 hour. Anti-MMP-1 antibody (Abcam, Cat. No.: ab137332, UK) was diluted to 1:1000 in 5% skim milk and reacted with the membrane for 2 hours. Subsequently, the membrane was washed three times with 0.1% PBS-T (0.1% Tween-20 in PBS solution) for 10 minutes each time. Goat anti-rabbit IgG (Jackson Immune Research, Cat. No.: 111-035-033, USA) was diluted to 1:3000 in 5% skim milk and reacted with the membrane for 2 hours. Subsequently, the membrane was detected using ECL solution (GE Healthcare, Cat. No.: RPN2232, USA). β-actin was identified using an anti-β-actin antibody (Santacruz biotechnology, USA) to compare the total amount of protein used in the experiment.

[0132] In addition, to analyze the amount of MMP-2, cell culture medium transferred to tubes was treated with 4X enzyme spectrum sample buffer to prepare samples, and the samples were SDS-PAGEd using an 8% SDS-PAGE gel. The gel was washed with 0.2% Tween-20 solution for 30 minutes, followed by washing with distilled water (DW) for 30 minutes. The gel was placed in TNCB buffer and incubated at 37°C for 16 hours. The treated gel was stained with Coomassie Brilliant Blue R250 (Sigma) and treated with destaining buffer (5% ethanol, 7.5% acetic acid, and distilled water). Subsequently, the activity of MMP-2 was confirmed by observing the empty bands on the gel that appeared during gelatin hydrolysis.

[0133] As a result, such Figure 6 As shown, it was confirmed that the peptide of the present invention inhibits the increased expression of MMP-1 and the activity of MMP-2 caused by ultraviolet irradiation.

[0134] Therefore, it can be concluded that the peptide containing the amino acid sequence of SEQ ID NO:1 reduces the expression of MMP-1 and the activity of MMP-2 in fibroblasts that are increased by ultraviolet radiation, thus inhibiting the aging process of cells and achieving the effect of reducing skin wrinkles.

[0135] 2-4. Confirmation of inhibition of MMP-1 expression increased by heat treatment

[0136] Infrared-induced skin thermal aging increases the expression of MMP-1, a protein encoding collagen degradation. Therefore, it was determined whether treatment with the peptides of this invention re-inhibited MMP-1 protein synthesis and whether cellular aging was suppressed.

[0137] First, NIH3T3 cells were loaded at 5 × 10⁻⁶. 5 Cells were seeded at a density of 10 cells / well in 6-well plates and then cultured in DMEM medium containing 10% fetal bovine serum (FBS) for 24 hours. The cultured cells were washed once with serum-free DMEM medium, and the peptides of the present invention were mixed in 1 mL of serum-free DMEM medium at concentrations of 10 µM, 50 µM, and 100 µM, respectively, and dispensed into the cells. Positive controls (PCs) were treated with 2.5 mM N-acetylcysteine ​​(NaC) instead of the peptides.

[0138] Cells were cultured in a CO2 incubator at 37°C for 24 hours. The top and bottom plates of a 6-well plate were sealed with paraffin membrane for heat treatment, and the bottom plate was immersed in a 44°C water bath for 40 minutes. After removing the culture medium, 1 mL of serum-free DMEM medium was added, and the plate was incubated in a CO2 incubator at 37°C for 8 hours. Subsequently, the cells were washed twice with PBS, and then cell lysis buffer was added to obtain lysates.

[0139] Samples were prepared by treating cells with 5X sample buffer, followed by SDS-PAGE using a 10% SDS-PAGE gel. Proteins separated by SDS-PAGE were transferred to a PVDF membrane. The membrane was blocked with 5% skim milk at room temperature for 1 hour. Anti-MMP-1 antibody (Abcam, Cat. No.: ab137332, UK) was diluted 1:1000 in 5% skim milk and reacted with the membrane for 2 hours. The membrane was washed three times with 0.1% PBS-T (PBS solution of 0.1% Tween-20) for 10 minutes each time. Goat anti-rabbit IgG (Jackson Immune Research, Cat. No.: 111-035-033, USA) was diluted 1:3000 in 5% skim milk and reacted with the membrane for 2 hours. Detection was performed using the membrane with ECL solution (GE Healthcare, Cat. No.: RPN2232, USA). Anti-β-actin antibody (Santacruz biotechnology, USA) was used to identify β-actin, thereby comparing the total amount of protein used in the experiment.

[0140] As a result, such Figure 7 As shown, the peptides of the present invention have been shown to reduce the secretion of MMP-1 protein, which is expressed in increased amounts in fibroblasts due to heat treatment.

[0141] Therefore, it can be concluded that the peptide containing the amino acid sequence of SEQ ID NO:1 causes the secretion of MMP-2 protein in fibroblasts, which was increased by heat treatment, to decrease and then recover, and the amount of collagen can be increased again. Thus, the cell aging phenomenon is inhibited, thereby achieving the effect of reducing skin wrinkles.

[0142] As described above, although the present invention has been described in detail with reference only to the embodiments described herein, it will be apparent to those skilled in the art that various modifications and alterations can be made without departing from the technical spirit of the present invention, and such modifications and alterations are obviously within the scope of the claims. <110> Kelgen Ltd. <120> Peptides with anti-aging activity and their uses <130> 2020-DPA-3975 <160> 13 <170> KoPatentIn 3.0 <210> 1 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> peptides <400> 1 Lys Arg Arg Arg Lys Arg Lys 1 5 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Col1a1-F <400> 2 caccctcaag agcctgagtc 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Col1a1-R <400> 3 agacggctga gtagggaaca 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Fibronectin-F <400> 4 ccaggaaccg agtacaccat 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Fibronectin-R <400> 5 atacccaggt tgggtgatga 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Elastin-F <400> 6 gcaagacctg gctttggact 20 <210> 7 <211> twenty one <212> DNA <213> Artificial Sequence <220> <223> Elastin-R <400> 7 gggagtttct ggttagggct g 21 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SIRT1-F <400> 8 tcagtggctg gaacagtgag 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> SIRT1-R <400> 9 tctggcatgt cccactatca 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> AQP3-F <400> 10 ccttcttggg tgctggaata 20 <210> 11 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> AQP3-R <400> 11 acacgataag ggaggctgtg 20 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH-F <400> 12 ggagccaaaa gggtcatcat 20 <210> 13 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> GAPDH-R <400> 13 gtgatggcat ggactgtggt 20

Claims

1. A peptide consisting of the amino acid sequence of SEQ ID NO:

1.

2. A composition comprising the peptide according to claim 1 as an active ingredient.

3. Use of the composition according to claim 2 in the preparation of a product for inhibiting skin aging.

4. Use of the composition according to claim 2 in the preparation of products for improving resistance to cell damage caused by aging or external stimuli.

5. The use according to claim 4, wherein the external stimulus is ultraviolet or infrared radiation.

6. The use according to claim 4, wherein the cells are skin fibroblasts or keratinocytes.

7. A cosmetic composition comprising the peptide according to claim 1 as an active ingredient.

8. Use of the cosmetic composition according to claim 7 in the preparation of a product for inhibiting skin aging.

9. Use of the peptide according to claim 1 in the preparation of a medicament for the prevention or treatment of photoaging.

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